Targeting vector, construction method for transgenic mouse capable of regulating and removing monocyte-derived DC via diphtheria toxin, and application of targeting vector
A technology for transgenic mice and targeting vectors, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, applications, etc.
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Embodiment 1
[0053] Example 1: Construction of a targeting vector with a long homology arm that targets the integration of the exogenous gene IRES-DTR into exon 7 of DC-SIGN.
[0054] The mouse DC-SIGN genome sequence was retrieved and downloaded from the genome.ucsc.edu website, combined with the mouse DC-SIGN (Genbank No: NM_133238.5) sequence, the positions and sequences of each exon and intron were determined. The BAC Clone containing the entire DC-SIGN gene: RP23-12K14 was purchased from the Children’hospotal Oakland Research Institute, and the BAC DNA was used HiPure Plasmid Filter Maxiprep Kit (Invitrogen) was prepared for use.
[0055] PCR amplification of the DC-SIGN 5' side homology arm (retrieval 5HA) and DC-SIGN 3' side homology arm (retrieval 3HA) of the target gene fragment: using RP23-12K14 BAC DNA as a template, using the forward primer DCSIGN-5HA -F(NotI)-ACCACC GCGGCCGC ACATCTGCCCATAGCACACAG (SEQ ID NO.1) and reverse primer DCSIGN-5HA-R (SpeI)-ACCACC ACTAGT AGCATCAG...
Embodiment 2
[0070] Example 2: Preparation of mouse embryonic stem (ES) cells targeted to insert IRES-DTR into DC-SIGN exon 7
[0071] Mouse embryonic fibroblasts (MEFs) culture: MEFs (ATCC) were cultured and maintained in MEF medium, passaged and frozen in time. MEF medium is DMEM medium with 10% FBS, 100U / ml penicillin streptomycin, 0.05mM 2mercaptoethanol, 2mML-glutamine.
[0072] Balb / c ES cell culture: MEFs cells were inactivated with 30 Gray γ-rays before use as trophoblast cells, Balb / c ES cells (Merck) were inoculated onto the inactivated MEFs, cultured and maintained with ES medium. ES cells were grown to 70% abundance and passaged at a ratio of 1:3. ES medium is DMEM medium with 15% FBS, 100U / ml penicillin, 1mM sodium pyruvate, 0.1mM non-essential amino acid, 0.05mM 2-mercaptoethanol, 2mM L-glutamine, 1μg / L leukemia inhibitory factor (LIF).
[0073] Electroporation of ES cells: ES cells were collected by trypsinization, washed once with PBS, resuspended in electroporation buff...
example 3
[0080] Example 3: Breeding DC SIGN-DTR transgenic mice with targeted insertion of IRES-DTR into DC-SIGN exon 7
[0081] Blastocyst injection of ES cells to obtain DC SIGN-DTR transgenic chimeric mice: Select 8-week-old well-developed C57BL / 6J male mice and C57BL / 6J female mice in a 1:2 cage, and select vaginal plug-positive females in the morning of the next day For mice, blastocysts can be obtained in the uterus if the female mouse is pregnant for 5 days. Ligated KM male mice and normal KM female mice were caged at a ratio of 1:2, and the female mice with vaginal plugs and swollen and ruddy vulva were selected as pseudopregnant KM female mice. The ES cells targeted to insert IRES-DTR to the DC-SIGN exon 7 site were injected into C57BL / 6J mouse blastocysts, and then inoculated into the uterus of pseudopregnant KM female mice, and chimeric mice were born ( F0), that is, cells derived from C57 and ES cells derived from BALB / c co-exist in the same individual, and the coat color ...
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